A taxonomy of transcriptomic cell types across the isocortex and hippocampal formation.

The isocortex and hippocampal formation (HPF) in the mammalian brain play critical roles in perception, cognition, emotion, and learning. We profiled ∼1.3 million cells covering the entire adult mouse isocortex and HPF and derived a transcriptomic cell-type taxonomy revealing a comprehensive repertoire of glutamatergic and GABAergic neuron types. Contrary to the traditional view of HPF as having a simpler cellular organization, we discover a complete set of glutamatergic types in HPF homologous to all major subclasses found in the six-layered isocortex, suggesting that HPF and the isocortex share a common circuit organization. We also identify large-scale continuous and graded variations of cell types along isocortical depth, across the isocortical sheet, and in multiple dimensions in hippocampus and subiculum. Overall, our study establishes a molecular architecture of the mammalian isocortex and hippocampal formation and begins to shed light on its underlying relationship with the development, evolution, connectivity, and function of these two brain structures.

A transcriptomic taxonomy of mouse brain-wide spinal projecting neurons.

The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.

A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain.

The mammalian brain consists of millions to billions of cells that are organized into many cell types with specific spatial distribution patterns and structural and functional properties1-3. Here we report a comprehensive and high-resolution transcriptomic and spatial cell-type atlas for the whole adult mouse brain. The cell-type atlas was created by combining a single-cell RNA-sequencing (scRNA-seq) dataset of around 7 million cells profiled (approximately 4.0 million cells passing quality control), and a spatial transcriptomic dataset of approximately 4.3 million cells using multiplexed error-robust fluorescence in situ hybridization (MERFISH). The atlas is hierarchically organized into 4 nested levels of classification: 34 classes, 338 subclasses, 1,201 supertypes and 5,322 clusters. We present an online platform, Allen Brain Cell Atlas, to visualize the mouse whole-brain cell-type atlas along with the single-cell RNA-sequencing and MERFISH datasets. We systematically analysed the neuronal and non-neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell-type organization in different brain regions-in particular, a dichotomy between the dorsal and ventral parts of the brain. The dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. Our study also uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types. Finally, we found that transcription factors are major determinants of cell-type classification and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole mouse brain transcriptomic and spatial cell-type atlas establishes a benchmark reference atlas and a foundational resource for integrative investigations of cellular and circuit function, development and evolution of the mammalian brain.

ATR activity controls stem cell quiescence via the cyclin F-SCF complex.

A key property of adult stem cells is their ability to persist in a quiescent state for prolonged periods of time. The quiescent state is thought to contribute to stem cell resilience by limiting accumulation of DNA replication­associated mutations. Moreover, cellular stress response factors are thought to play a role in maintaining quiescence and stem cell integrity. We utilized muscle stem cells (MuSCs) as a model of quiescent stem cells and find that the replication stress response protein, ATR (Ataxia Telangiectasia and Rad3-Related), is abundant and active in quiescent but not activated MuSCs. Concurrently, MuSCs display punctate RPA (replication protein A) and R-loop foci, both key triggers for ATR activation. To discern the role of ATR in MuSCs, we generated MuSC-specific ATR conditional knockout (ATRcKO) mice. Surprisingly, ATR ablation results in increased MuSC quiescence exit. Phosphoproteomic analysis of ATRcKO MuSCs reveals enrichment of phosphorylated cyclin F, a key component of the Skp1­Cul1­F-box protein (SCF) ubiquitin ligase complex and regulator of key cell-cycle transition factors, such as the E2F family of transcription factors. Knocking down cyclin F or inhibiting the SCF complex results in E2F1 accumulation and in MuSCs exiting quiescence, similar to ATR-deficient MuSCs. The loss of ATR could be counteracted by inhibiting casein kinase 2 (CK2), the kinase responsible for phosphorylating cyclin F. We propose a model in which MuSCs express cell-cycle progression factors but ATR, in coordination with the cyclin F­SCF complex, represses premature stem cell quiescence exit via ubiquitin­proteasome degradation of these factors.

Staufen1 inhibits MyoD translation to actively maintain muscle stem cell quiescence.

Tissue regeneration depends on the timely activation of adult stem cells. In skeletal muscle, the adult stem cells maintain a quiescent state and proliferate upon injury. We show that muscle stem cells (MuSCs) use direct translational repression to maintain the quiescent state. High-resolution single-molecule and single-cell analyses demonstrate that quiescent MuSCs express high levels of Myogenic Differentiation 1 (MyoD) transcript in vivo, whereas MyoD protein is absent. RNA pulldowns and costainings show that MyoD mRNA interacts with Staufen1, a potent regulator of mRNA localization, translation, and stability. Staufen1 prevents MyoD translation through its interaction with the MyoD 3'-UTR. MuSCs from Staufen1 heterozygous (Staufen1+/-) mice have increased MyoD protein expression, exit quiescence, and begin proliferating. Conversely, blocking MyoD translation maintains the quiescent phenotype. Collectively, our data show that MuSCs express MyoD mRNA and actively repress its translation to remain quiescent yet primed for activation.

The neonatal brain is not protected by osteopontin peptide treatment after hypoxia-ischemia.

Neonatal encephalopathy due to perinatal hypoxia-ischemia (HI) is a severe condition, and current treatment options are limited. Expression of endogenous osteopontin (OPN), a multifunction glycoprotein, is strongly upregulated in the brain after neonatal HI. Intracerebrally administered OPN has been shown to be neuroprotective following experimental neonatal HI and adult stroke. In the present study, we determined whether intranasal, intraperitoneal or intracerebral treatment with a smaller TAT-OPN peptide is neuroprotective in neonatal mice with HI brain damage. The TAT-OPN peptide exerts bioactivity as it was as potent as full-length OPN in inducing cell adhesion in an in vitro adhesion assay. Intranasal administration of TAT-OPN peptide immediately after HI (T0) or in a repetitive treatment schedule of T0, 3 h, day (D) 1, 2 and 3 after HI did not protect cerebral gray or white matter after HI. Intraperitoneal TAT-OPN treatment at T0 or in two extended treatment schedules (D5, 7, 9, 11, 13, 15 after HI or T0, D1, 3, 5, 7, 9, 11, 13 and 15 after HI) did not result in neuroprotection either. Moreover, no functional improvement (cylinder rearing test and adhesive removal task) was observed following TAT-OPN treatment in any of the intraperitoneal treatment schedules. We validated that the TAT-OPN peptide reached the brain after intranasal or intraperitoneal administration by using an HIV-TAT staining. Finally, also intracerebral administration of the TAT-OPN peptide 1 h after HI did not reduce cerebral damage. Our data show that administration of the TAT-OPN peptide did not exert neuroprotective effects on neonatal HI-induced brain injury or sensorimotor behavioral deficits.

Assessment of long-term safety and efficacy of intranasal mesenchymal stem cell treatment for neonatal brain injury in the mouse.

BACKGROUND: For clinical translation, we assessed whether intranasal mesenchymal stem cell (MSC) treatment after hypoxia-ischemia (HI) induces neoplasia in the brain or periphery at 14 mo. Furthermore, the long-term effects of MSCs on behavior and lesion size were determined. METHOD: HI was induced in 9-d-old mice. Pups received an intranasal administration of 0.5 × 10(6) MSCs or vehicle at 10 d post-HI. Full macroscopical and microscopical pathological analysis of 39 organs per mouse was performed. Sensorimotor behavior was assessed in the cylinder-rearing test at 10 d, 28 d, 6 mo, and 9 mo. Cognition was measured with the novel object recognition test at 3 and 14 mo post-HI. Lesion size was determined by analyzing mouse-anti-microtubule-associated protein 2 (MAP2) and mouse-anti-myelin basic protein (MBP) staining at 5 wk and 14 mo. RESULTS: At 14 mo post-HI, we did not observe any neoplasia in the nasal turbinates, brain, or other organs of HI mice treated with MSCs. Furthermore, our results show that MSC-induced improvement of sensorimotor and cognitive function is long lasting. In contrast, HI-vehicle mice showed severe behavioral impairment. Recovery of MAP2- and MBP-positive area lasted up to 14 mo following MSC treatment. CONCLUSION: Our results provide strong evidence of the long-term safety and positive effects of MSC treatment following neonatal HI in mice.

Rabies virus-based barcoded neuroanatomy resolved by single-cell RNA and in situ sequencing.

Mapping the connectivity of diverse neuronal types provides the foundation for understanding the structure and function of neural circuits. High-throughput and low-cost neuroanatomical techniques based on RNA barcode sequencing have the potential to map circuits at cellular resolution and a brain-wide scale, but existing Sindbis virus-based techniques can only map long-range projections using anterograde tracing approaches. Rabies virus can complement anterograde tracing approaches by enabling either retrograde labeling of projection neurons or monosynaptic tracing of direct inputs to genetically targeted postsynaptic neurons. However, barcoded rabies virus has so far been only used to map non-neuronal cellular interactions in vivo and synaptic connectivity of cultured neurons. Here we combine barcoded rabies virus with single-cell and in situ sequencing to perform retrograde labeling and transsynaptic labeling in the mouse brain. We sequenced 96 retrogradely labeled cells and 295 transsynaptically labeled cells using single-cell RNA-seq, and 4130 retrogradely labeled cells and 2914 transsynaptically labeled cells in situ. We found that the transcriptomic identities of rabies virus-infected cells can be robustly identified using both single-cell RNA-seq and in situ sequencing. By associating gene expression with connectivity inferred from barcode sequencing, we distinguished long-range projecting cortical cell types from multiple cortical areas and identified cell types with converging or diverging synaptic connectivity. Combining in situ sequencing with barcoded rabies virus complements existing sequencing-based neuroanatomical techniques and provides a potential path for mapping synaptic connectivity of neuronal types at scale.

In the brain, messages are relayed from one cell to the next through intricate networks of axons and dendrites that physically interact at junctions known as synapses. Mapping out this synaptic connectivity ­ that is, exactly which neurons are connected via synapses ­ remains a major challenge. Monosynaptic tracing is a powerful approach that allows neuroscientists to explore neural networks by harnessing viruses which spread between neurons via synapses, in particular the rabies virus. This pathogen travels exclusively from 'postsynaptic' to 'presynaptic' neurons ­ from the cell that receives a message at a synapse, back to the one that sends it. A modified variant of the rabies virus can therefore be used to reveal the presynaptic cells connecting to a population of neurons in which it has been originally introduced. However, this method does not allow scientists to identify the exact postsynaptic neuron that each presynaptic cell is connected to. One way to bypass this issue is to combine monosynaptic tracing with RNA barcoding to create distinct versions of the modified rabies virus, which are then introduced into separate populations of neurons. Tracking the spread of each version allows neuroscientists to spot exactly which presynaptic cells signal to each postsynaptic neuron. So far, this approach has been used to examine synaptic connectivity in neurons grown in the laboratory, but it remains difficult to apply it to neurons in the brain. In response, Zhang, Jin et al. aimed to demonstrate how monosynaptic tracing that relies on barcoded rabies viruses could be used to dissect neural networks in the mouse brain. First, they confirmed that it was possible to accurately detect which version of the virus had spread to presynaptic neurons using both in situ and single-cell RNA sequencing. Next, they described how this information could be analysed to build models of potential neural networks, and what type of additional experiments are required for this work. Finally, they used the approach to identify neurons that tend to connect to the same postsynaptic cells and then investigated what these have in common, showing how the technique enables a finer understanding of neural circuits. Overall, the work by Zhang, Jin et al. provides a comprehensive review of the requirements and limitations associated with monosynaptic tracing experiments based on barcoded rabies viruses, as well as how the approach could be optimized in the future. This information will be of broad interest to scientists interested in mapping neural networks in the brain.

The transcriptomic and spatial organization of telencephalic GABAergic neuronal types.

The telencephalon of the mammalian brain comprises multiple regions and circuit pathways that play adaptive and integrative roles in a variety of brain functions. There is a wide array of GABAergic neurons in the telencephalon; they play a multitude of circuit functions, and dysfunction of these neurons has been implicated in diverse brain disorders. In this study, we conducted a systematic and in-depth analysis of the transcriptomic and spatial organization of GABAergic neuronal types in all regions of the mouse telencephalon and their developmental origins. This was accomplished by utilizing 611,423 single-cell transcriptomes from the comprehensive and high-resolution transcriptomic and spatial cell type atlas for the adult whole mouse brain we have generated, supplemented with an additional single-cell RNA-sequencing dataset containing 99,438 high-quality single-cell transcriptomes collected from the pre- and postnatal developing mouse brain. We present a hierarchically organized adult telencephalic GABAergic neuronal cell type taxonomy of 7 classes, 52 subclasses, 284 supertypes, and 1,051 clusters, as well as a corresponding developmental taxonomy of 450 clusters across different ages. Detailed charting efforts reveal extraordinary complexity where relationships among cell types reflect both spatial locations and developmental origins. Transcriptomically and developmentally related cell types can often be found in distant and diverse brain regions indicating that long-distance migration and dispersion is a common characteristic of nearly all classes of telencephalic GABAergic neurons. Additionally, we find various spatial dimensions of both discrete and continuous variations among related cell types that are correlated with gene expression gradients. Lastly, we find that cortical, striatal and some pallidal GABAergic neurons undergo extensive postnatal diversification, whereas septal and most pallidal GABAergic neuronal types emerge simultaneously during the embryonic stage with limited postnatal diversification. Overall, the telencephalic GABAergic cell type taxonomy can serve as a foundational reference for molecular, structural and functional studies of cell types and circuits by the entire community.

Mesenchymal stem cell transplantation attenuates brain injury after neonatal stroke.

BACKGROUND AND PURPOSE: Brain injury caused by stroke is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. Mesenchymal stem cells (MSC) have been shown to improve outcome after neonatal hypoxic-ischemic brain injury mainly by secretion of growth factors stimulating repair processes. We investigated whether MSC treatment improves recovery after neonatal stroke and whether MSC overexpressing brain-derived neurotrophic factor (MSC-BDNF) further enhances recovery. METHODS: We performed 1.5-hour transient middle cerebral artery occlusion in 10-day-old rats. Three days after reperfusion, pups with evidence of injury by diffusion-weighted MRI were treated intranasally with MSC, MSC-BDNF, or vehicle. To determine the effect of MSC treatment, brain damage, sensorimotor function, and cerebral cell proliferation were analyzed. RESULTS: Intranasal delivery of MSC- and MSC-BDNF significantly reduced infarct size and gray matter loss in comparison with vehicle-treated rats without any significant difference between MSC- and MSC-BDNF-treatment. Treatment with MSC-BDNF significantly reduced white matter loss with no significant difference between MSC- and MSC-BDNF-treatment. Motor deficits were also improved by MSC treatment when compared with vehicle-treated rats. MSC-BDNF-treatment resulted in an additional significant improvement of motor deficits 14 days after middle cerebral artery occlusion, but there was no significant difference between MSC or MSC-BDNF 28 days after middle cerebral artery occlusion. Furthermore, treatment with either MSC or MSC-BDNF induced long-lasting cell proliferation in the ischemic hemisphere. CONCLUSIONS: Intranasal administration of MSC after neonatal stroke is a promising therapy for treatment of neonatal stroke. In this experimental paradigm, MSC- and BNDF-hypersecreting MSC are equally effective in reducing ischemic brain damage.

Mesenchymal stem cells restore cortical rewiring after neonatal ischemia in mice.

OBJECTIVE: A study was undertaken to investigate the effect of neonatal hypoxic-ischemic (HI) brain damage and mesenchymal stem cell (MSC) treatment on the structure and contralesional connectivity of motor function-related cerebral areas. METHODS: Brain remodeling after HI±MSC treatment in neonatal mice was analyzed using diffusion tensor magnetic resonance imaging, immunohistochemistry, anterograde tracing with biotinylated dextran amine (BDA), and retrograde tracing with fluorescent pseudorabies virus (PRV). RESULTS: MSC treatment after HI reduced contralesional rewiring taking place after HI. Following MSC treatment, fractional anisotropy values, which were increased in both ipsi- and contralesional cortices and decreased in the corpus callosum (CC) after HI, were normalized to the level observed in sham-operated mice. These results were corroborated by myelin basic protein intensity and staining pattern in these areas. Anterograde tracing of ipsilesional motor neurons showed that after MSC treatment, fewer BDA-positive fibers crossed the CC and extended into the contralesional motor cortex compared to HI mice. This remodeling was functional, because retrograde labeling showed increased connectivity between impaired (left) forepaw and the contralesional (left) motor cortex after HI, whereas MSC treatment reduced this connection and increased the connection between the impaired (left) forepaw and the ipsilesional (right) motor cortex. Finally, the extent of contralesional rewiring measured with BDA and PRV tracing was related to sensorimotor dysfunction. INTERPRETATION: This is the first study to describe MSC treatment after neonatal HI markedly reducing contralesional axonal remodeling induced by HI brain injury.

Cell-type specific molecular signatures of aging revealed in a brain-wide transcriptomic cell-type atlas.

Biological aging can be defined as a gradual loss of homeostasis across various aspects of molecular and cellular function. Aging is a complex and dynamic process which influences distinct cell types in a myriad of ways. The cellular architecture of the mammalian brain is heterogeneous and diverse, making it challenging to identify precise areas and cell types of the brain that are more susceptible to aging than others. Here, we present a high-resolution single-cell RNA sequencing dataset containing ~1.2 million high-quality single-cell transcriptomic profiles of brain cells from young adult and aged mice across both sexes, including areas spanning the forebrain, midbrain, and hindbrain. We find age-associated gene expression signatures across nearly all 130+ neuronal and non-neuronal cell subclasses we identified. We detect the greatest gene expression changes in non-neuronal cell types, suggesting that different cell types in the brain vary in their susceptibility to aging. We identify specific, age-enriched clusters within specific glial, vascular, and immune cell types from both cortical and subcortical regions of the brain, and specific gene expression changes associated with cell senescence, inflammation, decrease in new myelination, and decreased vasculature integrity. We also identify genes with expression changes across multiple cell subclasses, pointing to certain mechanisms of aging that may occur across wide regions or broad cell types of the brain. Finally, we discover the greatest gene expression changes in cell types localized to the third ventricle of the hypothalamus, including tanycytes, ependymal cells, and Tbx3+ neurons found in the arcuate nucleus that are part of the neuronal circuits regulating food intake and energy homeostasis. These findings suggest that the area surrounding the third ventricle in the hypothalamus may be a hub for aging in the mouse brain. Overall, we reveal a dynamic landscape of cell-type-specific transcriptomic changes in the brain associated with normal aging that will serve as a foundation for the investigation of functional changes in the aging process and the interaction of aging and diseases.

A high-resolution transcriptomic and spatial atlas of cell types in the whole mouse brain.

The mammalian brain is composed of millions to billions of cells that are organized into numerous cell types with specific spatial distribution patterns and structural and functional properties. An essential step towards understanding brain function is to obtain a parts list, i.e., a catalog of cell types, of the brain. Here, we report a comprehensive and high-resolution transcriptomic and spatial cell type atlas for the whole adult mouse brain. The cell type atlas was created based on the combination of two single-cell-level, whole-brain-scale datasets: a single-cell RNA-sequencing (scRNA-seq) dataset of ~7 million cells profiled, and a spatially resolved transcriptomic dataset of ~4.3 million cells using MERFISH. The atlas is hierarchically organized into five nested levels of classification: 7 divisions, 32 classes, 306 subclasses, 1,045 supertypes and 5,200 clusters. We systematically analyzed the neuronal, non-neuronal, and immature neuronal cell types across the brain and identified a high degree of correspondence between transcriptomic identity and spatial specificity for each cell type. The results reveal unique features of cell type organization in different brain regions, in particular, a dichotomy between the dorsal and ventral parts of the brain: the dorsal part contains relatively fewer yet highly divergent neuronal types, whereas the ventral part contains more numerous neuronal types that are more closely related to each other. We also systematically characterized cell-type specific expression of neurotransmitters, neuropeptides, and transcription factors. The study uncovered extraordinary diversity and heterogeneity in neurotransmitter and neuropeptide expression and co-expression patterns in different cell types across the brain, suggesting they mediate a myriad of modes of intercellular communications. Finally, we found that transcription factors are major determinants of cell type classification in the adult mouse brain and identified a combinatorial transcription factor code that defines cell types across all parts of the brain. The whole-mouse-brain transcriptomic and spatial cell type atlas establishes a benchmark reference atlas and a foundational resource for deep and integrative investigations of cell type and circuit function, development, and evolution of the mammalian brain.

Targeting the p53 pathway to protect the neonatal ischemic brain.

OBJECTIVE: To investigate whether inhibition of mitochondrial p53 association using pifithrin-µ (PFT-µ) represents a potential novel neuroprotective strategy to combat perinatal hypoxic-ischemic (HI) brain damage. METHODS: Seven-day-old rats were subjected to unilateral carotid artery occlusion and hypoxia followed by intraperitoneal treatment with PFT-µ, an inhibitor of p53 mitochondrial association or PFT-α an inhibitor of p53 transcriptional activity. Cerebral damage, sensorimotor and cognitive function, apoptotic pathways (cytosolic cytochrome c, Smac/DIABLO, active caspase 3), and oxidative stress (lipid peroxidation and PARP-1 cleavage) were investigated. RESULTS: PFT-µ treatment completely prevented the HI-induced increase in mitochondrial p53 association at 3 hours and reduced neuronal damage at 48 hours post-HI. PFT-µ had long-term (6-10 weeks post-HI) beneficial effects as sensorimotor and cognitive outcome improved and infarct size was reduced by ~79%. Neuroprotection by PFT-µ treatment was associated with strong inhibition of apoptotic pathways and reduced oxidative stress. Unexpectedly, PFT-µ also inhibited HI-induced upregulation of p53 target genes. However, the neuroprotective effect of inhibiting only p53 transcriptional activity by PFT-α was significantly smaller and did not involve reduced oxidative stress. INTERPRETATION: We are the first to show that prevention of mitochondrial p53 association by PFT-µ strongly improves functional outcome and decreases lesion size after neonatal HI. PFT-µ not only inhibits mitochondrial release of cytochrome c, but also inhibits oxidative stress. We propose that as a consequence nuclear accumulation of p53 and transcription of proapoptotic target genes are prevented. In conclusion, targeting p53 mitochondrial association by PFT-µ may develop into a novel and powerful neuroprotective strategy.

Mesenchymal stem cells as a treatment for neonatal ischemic brain damage.

Mesenchymal stem cell (MSC)-based therapies have been proven effective in experimental models of numerous disorders. Treatment of ischemic brain injury by transplantation of MSCs in neonatal animal models has been shown to be effective in reducing lesion volume and improving functional outcome. The beneficial effect of MSC transplantation to treat neonatal brain injury might be explained by the great plasticity of the neonatal brain. The neonatal brain is still in a developmentally active phase, leading to a better efficiency of MSC transplantation than that observed in experiments using adult models of stroke. Enhanced neurogenesis and axonal remodeling likely underlie the improved functional outcome following MSC treatment after neonatal hypoxic-ischemic (HI) brain injury. With respect to the mechanism of repair by MSCs, MSCs do not survive long term and replace damaged tissue themselves. We propose that MSCs react to the needs of the ischemic cerebral environment by secretion of several growth factors, cytokines, and other bioactive molecules to regulate damage and repair processes. Parenchymal cells react to the secretome of the MSCs and contribute to stimulate repair processes. These intrinsic adaptive properties of MSCs make them excellent candidates for a novel therapy to treat the devastating effects of HI encephalopathy in the human neonate.

Repeated mesenchymal stem cell treatment after neonatal hypoxia-ischemia has distinct effects on formation and maturation of new neurons and oligodendrocytes leading to restoration of damage, corticospinal motor tract activity, and sensorimotor function.

Birth asphyxia is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. We show that a single mesenchymal stem cell treatment at 3 d (MSC-3) after neonatal hypoxia-ischemia (HI) in postnatal day 9 mice improved sensorimotor function and reduced lesion size. A second MSC treatment at 10 d after HI (MSC-3+10) further enhanced sensorimotor improvement and recovery of MAP2 and MBP (myelin basic protein) staining. Ipsilateral anterograde corticospinal tract tracing with biotinylated dextran amine (BDA) showed that HI reduced BDA labeling of the contralateral spinal cord. Only MSC-3+10 treatment partially restored contralateral spinal cord BDA staining, indicating enhanced axonal remodeling. MSC-3 enhanced formation of bromodeoxyuridine-positive neurons and oligodendrocytes. Interestingly, the second gift at day 10 did not further increase new cell formation, whereas only MSC-10 did. These findings indicate that increased positive effect of MSC-3+10 compared with MSC-3 alone is mediated via distinct pathways. We hypothesize that MSCs adapt their growth and differentiation factor production to the needs of the environment at the time of intracranial injection. Comparing the response of MSCs to in vitro culture with HI brain extracts obtained at day 10 from MSC-3- or vehicle-treated animals by pathway-focused PCR array analysis revealed that 29 genes encoding secreted factors were indeed differentially regulated. We propose that the function of MSCs is dictated by adaptive specific signals provided by the damaged and regenerating brain.

GRK2: a novel cell-specific regulator of severity and duration of inflammatory pain.

Chronic pain associated with inflammation is a common clinical problem, and the underlying mechanisms have only begun to be unraveled. GRK2 regulates cellular signaling by promoting G-protein-coupled receptor (GPCR) desensitization and direct interaction with downstream kinases including p38. The aim of this study was to determine the contribution of GRK2 to regulation of inflammatory pain and to unravel the underlying mechanism. GRK2(+/-) mice with an approximately 50% reduction in GRK2 developed increased and markedly prolonged thermal hyperalgesia and mechanical allodynia after carrageenan-induced paw inflammation or after intraplantar injection of the GPCR-binding chemokine CCL3. The effect of reduced GRK2 in specific cells was investigated using Cre-Lox technology. Carrageenan- or CCL3-induced hyperalgesia was increased but not prolonged in mice with decreased GRK2 only in Na(v)1.8 nociceptors. In vitro, reduced neuronal GRK2 enhanced CCL3-induced TRPV1 sensitization. In vivo, CCL3-induced acute hyperalgesia in GRK2(+/-) mice was mediated via TRPV1. Reduced GRK2 in microglia/monocytes only was required and sufficient to transform acute carrageenan- or CCL3-induced hyperalgesia into chronic hyperalgesia. Chronic hyperalgesia in GRK2(+/-) mice was associated with ongoing microglial activation and increased phospho-p38 and tumor necrosis factor alpha (TNF-alpha) in the spinal cord. Inhibition of spinal cord microglial, p38, or TNF-alpha activity by intrathecal administration of specific inhibitors reversed ongoing hyperalgesia in GRK2(+/-) mice. Microglia/macrophage GRK2 expression was reduced in the lumbar ipsilateral spinal cord during neuropathic pain, underlining the pathophysiological relevance of microglial GRK2. Thus, we identified completely novel cell-specific roles of GRK2 in regulating acute and chronic inflammatory hyperalgesia.

Osteopontin enhances endogenous repair after neonatal hypoxic-ischemic brain injury.

BACKGROUND AND PURPOSE: Hypoxic-ischemic (HI) brain injury is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. To identify molecules important for cerebral damage and repair, we investigated the growth factor-related gene expression profile after neonatal cerebral HI. We identified osteopontin (OPN) as the most highly upregulated factor early after HI. We therefore explored the role of endogenous OPN in brain damage and repair. METHODS: Nine-day-old wild-type mice were exposed to cerebral HI; growth factor-related gene expression profiles were analyzed 1 to 7 days later by reverse transcriptase-polymerase chain reaction arrays. To determine the contribution of OPN to brain damage, we used p9 OPN(-/-) and wild-type mice. HI brain damage, sensorimotor function, and cell proliferation and differentiation were compared. RESULTS: Gene expression profiling of 150 genes related to growth factors and neurotrophins showed that expression of 52 genes changed during the first 7 days after HI. OPN was the gene with the strongest increase expression at all time points measured. We show here for the first time that in response to neonatal HI, OPN-deficient mice developed increased gray and white matter loss and more pronounced sensorimotor deficits as compared with wild-type littermates. Furthermore, OPN deficiency decreases HI-induced cell proliferation/survival and oligodendrogenesis without affecting neuronal differentiation. CONCLUSIONS: OPN plays an important role in repairing brain injury after neonatal HI by regulating cerebral cell proliferation/survival and oligodendrocyte differentiation after injury. The observed promyelinative effect of OPN may offer novel possibilities for a therapy targeting white matter injury.

Mesenchymal stem cell transplantation changes the gene expression profile of the neonatal ischemic brain.

Mesenchymal stem cell (MSC) treatment is an effective strategy to reduce brain damage after neonatal hypoxia-ischemia (HI) in mice. We recently showed that a single injection with MSC at either 3 or 10 days after HI (MSC-3 or MSC-10) increases neurogenesis. In case of two injections (MSC-3+10), the second MSC application does not increase neurogenesis, but promotes corticospinal tract remodeling. Here we investigated GFP(+)-MSC engraftment level in the brain using quantitative-PCR analysis. We show for the first time that in the neonatal ischemic brain survival of transplanted MSC is very limited. At 3 days after injection ∼22% of transplanted MSC were still detectable and 18 days after the last administration barely ∼1%. These findings indicate that engraftment of MSC is not likely the underlying mechanism of the efficient regenerative process. Therefore, we tested the hypothesis that the effects of MSC-treatment on regenerative processes are related to specific changes in the gene expression of growth factors and cytokines in the damaged area of the brain using PCR-array analysis. We compared the effect of one (MSC-10) or two (MSC-3+10) injections of 10(5) MSC on gene expression in the brain. Our data show that MSC-10 induced expression of genes regulating proliferation/survival. In response to MSC-3+10-treatment a pattern functionally categorized as growth stimulating genes was increased. Collectively, our data indicate that specific regulation of the endogenous growth factor milieu rather than replacement of damaged tissue by exogenous MSC mediates regeneration of the damaged neonatal brain by MSC-treatment.

Regional, Layer, and Cell-Type-Specific Connectivity of the Mouse Default Mode Network.

The evolutionarily conserved default mode network (DMN) is a distributed set of brain regions coactivated during resting states that is vulnerable to brain disorders. How disease affects the DMN is unknown, but detailed anatomical descriptions could provide clues. Mice offer an opportunity to investigate structural connectivity of the DMN across spatial scales with cell-type resolution. We co-registered maps from functional magnetic resonance imaging and axonal tracing experiments into the 3D Allen mouse brain reference atlas. We find that the mouse DMN consists of preferentially interconnected cortical regions. As a population, DMN layer 2/3 (L2/3) neurons project almost exclusively to other DMN regions, whereas L5 neurons project in and out of the DMN. In the retrosplenial cortex, a core DMN region, we identify two L5 projection types differentiated by in- or out-DMN targets, laminar position, and gene expression. These results provide a multi-scale description of the anatomical correlates of the mouse DMN.